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Drug-Drug Interactions: Inhibition and Induction. Michael W. Sinz, Ph.D. Pharmaceutical Candidate Optimization Metabolism and Pharmacokinetics Bristol Myers Squibb. Pharmaceutical Research Institute Wallingford, CT [email protected] Drug Development Process: Discovery-Approval. PI.

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slide1

Drug-Drug Interactions:

Inhibition and Induction

Michael W. Sinz, Ph.D.

Pharmaceutical Candidate Optimization

Metabolism and Pharmacokinetics

Bristol Myers Squibb

Pharmaceutical Research Institute

Wallingford, CT

[email protected]

slide2

Drug Development Process: Discovery-Approval

PI

P2

P3

Discovery Preclinical Clinical FDA

Approval

Time (yr): 4 2 1.5 2 3.5 1

#’s: 30,000 2000 200 40 12 8

  • Drug Development Process-
  • 10-15 years
  • 500-800 million dollars
  • 0.003% chance of a return on investment (1/30,000)

C&EN, 1/28/02, KJ Watkins and DDT 6(18), 2001 Shillingford and Vose

drug metabolizing enzymes
Liver is the major organ for drug metabolism / elimination

Phase I and Phase II Enzymes

Phase I: oxidative or hydrolytic reactions

Phase II: conjugative reactions

Predominate enzyme system that metabolizes drugs is the cytochrome P450 (CYP450) family of enzymes which mediate oxidation reactions, such as hydroxylations

Drug Metabolizing Enzymes

Proportions of CYP450 Enzymes

In Human Liver

Other CYPs

3A4

2D6

2A6

2E1

1A2

2C9/19

model systems to study drug interactions

Complexity

Confidence

Speed

Simplicity

Model Systems to Study Drug Interactions
  • In Vitro Systems
    • cDNA expressed enzymes (rCYP’s)
    • microsomes (subcellular fraction of ER)
    • hepatocytes (primary cultures)
  • In Vivo Systems
    • animals (mouse, rat, dog, monkey, transgenics)
    • humans (volunteers, patients)
slide5

Drug Development Process: Discovery-Approval

PI

P2

P3

Discovery Preclinical Clinical FDA

Approval

How do we improve the

process and increase the

success rate?

Time (yr): 4 2 1.5 2 3.5 1

#’s: 30,000 2000 200 40 12 8

  • Drug Development Process-
  • 10-15 years
  • 500-800 million dollars
  • 0.003% chance of a return on investment (1/30,000)

C&EN, 1/28/02, KJ Watkins and DDT 6(18), 2001 Shillingford and Vose

metabolic drug interactions

Activity

Drug Conc.

Activity

Drug Conc.

Metabolic Drug Interactions
  • Inhibition
  • Induction
  • Polymorphism (CYP2D6)
  • Formation of reactive, toxic, or active metabolites
  • Disease state
slide7

Examples of “Undesirable” Drugs

Mibefradil (Posicor)

> Cytochrome P450 3A4 (CYP3A4) inhibitor

Terfenadine (Seldane)

> Extensive metabolism (primarily CYP3A4)

Withdrawn

Cisapride (Propulsid)

> QT prolongation

Astemizole (Hismanal)

Troglitazone (Rezulin)

> Hepatotoxic

> Metabolism to reactive intermediates

Ritonavir (Norvir)

> Potent CYP3A4 inhibitor

> Potent P-glycoprotein inhibitor

> Broad spectrum inducer

Recognized issue with regulatory agencies and the pharmaceutical industry.

Predict early and eliminate such compounds to avoid safety issues, regulatory obstacles, and market pressures.

not all drug interactions are bad
Not All Drug Interactions Are Bad

The use of a cyclosporin–ketoconazole combination: making renal transplantation affordable in developing countries.

T. Gerntholtz, M. D. Pascoe, J. F. Botha, J. Halkett and D. Kahn. Eur J Clin Pharmacol (2004)

Pharmacokinetic enhancement of protease inhibitor therapy; Ritonavir-saquinavir; ritonavir-lopinavir

King JR, Wynn H, Brundage R, Acosta EP. Clin Pharmacokinet (2004)

slide9

CYP450 - Mediated Interactions

CYP450 Inhibition

Reversible Inhibition

Irreversible Inhibition

slide10

Reversible vs Irreversible Inhibition

True

Irreversible

Reversible

Quasi-

Irreversible

Metabolite

Metabolite

Metabolite

Fe

Fe

Fe

cyp inhibition models and analytical methods

discovery

preclinical

clinical

CYP Inhibition: Models and Analytical Methods

rCYP &

flourescent

probes

microsomes &

“drug probes”

patients &

drug probes

Automated liquid handlers

Fluorescent plate readers

Automated data analysis

Automated liquid handlers or not

FL plate readers, LC-UV / FL, LC-MS

Probe-Drug Metabolite

Probe-Drug + Test Compound Metabolite

IC50

or Ki

how to employ cyp inhibition

discovery

preclinical

clinical

How to Employ CYP Inhibition

IC50 Determination

KiDetermination

Change in AUC

Eliminate potent inhibitors

Rank order compounds

Characterize inhibition

Predict interaction potential

Assess changes in PK

- increase in AUC

slide13

Semi-Quantitative Predictions of Drug Interactions

Relationship between in vitro Ki and plasma concentration

of the inhibitor.

Generally accepted guideline for evaluating risk by PhRMA

and regulatory agencies.

[I]/Ki > 1.0 (interaction “likely”)

[I]/Ki = 0.1 to 1.0 (interaction “possible”)

[I]/Ki < 0.1 (interaction “remote”)

[I] = Plasma Cmax,total (free and bound)

Bjornsson, et al. DMD (2003) and Tucker, et al. Pharm.Res. (2001)

measurement of plasma liver concentration

Cmax

Concentration

Time

Measurement of Plasma (Liver) Concentration

Estimate liver concentration

by measuring systemic plasma

concentrations.

CYP450

Biliary elimination

Metabolism

Drug concentrating in cells

slide15

Reversible vs Irreversible Inhibition

True

Irreversible

Reversible

Quasi-

Irreversible

Metabolite

Metabolite

Metabolite

Fe

Fe

Fe

slide16

Duration of Inhibitory Effects

Inhibitory Effect

Inhibitory Effect

Conc. of Drug

Conc. of Drug

Time

Time

Reversible Enzyme Inhibition Irreversible Enzyme Inhibition

Inhibition effect extends beyond elimination of drug due to enzyme inactivation.

Effect tends to accumulate after each dose.

Inhibition effect is generally greater than predicted based on ‘reversible’ IC50 or Ki values.

Most compounds will have non-linear pharmacokinetics.

Rare cases of hepatotoxicity associated with covalently bound adducts.

More difficult to predict inhibitory effects in patients.

slide17

Examples of Reversible & Irreversible Inhibitors

  • Irreversible Inhibitors
  • Posicor

removed from the market due to CYP3A4 interactions

major drug interactions, 2-10X changes in pharmacokinetics

  • Clarithromycin, Troleandomycin, Erythromycin

older drugs - irreversible inhibition was not understood

moderate drug interactions (3A4), 2-6X changes in pharmacokinetics

  • Ritonavir

black box warning due to drug interactions

major drug interactions (3A4), 2-50X changes in pharmacokinetics

Reversible Inhibitors

  • Ketoconazole

major drug interactions (3A4), 100X changes in pharmacokinetics

  • Quinidine, Paroxetine, Fluoxetine

major drug interactions (2D6)

slide19

CYP450 - Mediated Interactions

CYP450 Induction

Induction

Autoinduction

percent reduction in auc s due to cyp3a4 enzyme induction
Percent Reduction in AUC’s Due to CYP3A4 Enzyme Induction

Increased elimination

of drugs and loss of

efficacy

Indinavir and St Johns Wort

Carbamazepine

Conc. of Drug

Conc. of Drug

loss of

efficacy

loss of

efficacy

Time (days)

Time (hr)

cyp induction models and analytical methods

discovery

preclinical

clinical

CYP Induction: Models and Analytical Methods

Patients

Receptor binding

Cell based transactivation

Immortalized cells

Hepatocytes

Immortalized cells

Transgenic animals

Luminescence

RT-PCR

Enzyme activity (LC-MS)

Western blotting

RT-PCR

Changes in

pharmacokinetics

LC-MS

Probe-Drug Metabolite

Probe-Drug + Test Compound Metabolite

Fold increase in activity

nuclear hormone receptors involved in enzyme induction of cyp450 s
Nuclear Hormone Receptors Involved inEnzyme Induction of CYP450’s

Major mechanism of enzyme induction involves increased

transcription of P450 by NHR’s.

Minor mechanisms of induction include mRNA and

protein stabilization (ie., longer half-life). Example: CYP2E1

pxr mediated induction of cyp3a4

RXR

PXR

Transcription

CYP3A4 mRNA

CYP3A4

Drug-OH

Drug

PXR Mediated Induction of CYP3A4

L

SRC-1

RNA poly II

TFs

Translation

PXR

response

element

Promoter

CYP3A4 gene

Key Events:

Ligand Binding

Complex Activation

Gene Transcription

mRNA Translation

= Increased Enzyme Activity

pxr transactivation assay

REPORTER

PXR Transactivation Assay

PXR

PXR

RXR

Cyp3A4 promoter

(Luciferase)

HepG2 cells

slide25

Primary Culture of Human Hepatocytes

Drug treatment for 3-5 days in culture.

Proteins and RNA extracted and

analyzed by Western blotting,

enzyme activity, and/or RT-PCR.

ECM

Hepatocytes

slide26

CYP3A4

CYP1A2

7-Ethoxyresorufin O-dealkylation

(pmol/mg protein/min)

Testosterone 6b-hydroxylation

(pmol/mg protein/min)

9

9

anti-CYP1A

anti-CYP3A

CYP2C9

CYP2B6

7-EFC O-deethylation

(pmol/mg protein/min)

Tolbutamide methylhydroxylation

(pmol/mg protein/min)

9

anti-CYP2B

1 = CON, 2 = RIF, 3 = PB, 4 = CLF, 5 = PCN, 6 = MPN, 7 = OMP, 8 = PHN

knock out and transgenic pxr mice
Knock Out and Transgenic PXR Mice

hPXR

mPXR

Potential model to bridge in vitro and in vivo data

Still a mouse with a single gene change!

animal models of human induction species differences
Animal Models of Human Induction?Species Differences

PXR

  • Rezulin
    • potent human inducer
    • no induction in rats
  • Rifampicin
    • potent inducer in humans and rabbits
    • weak inducer in rodents
  • Pregnenolone 16-alpha Carbonitrile
    • potent inducer in rodents
    • weak inducer in humans
  • Phenobarbital
    • fairly equal induction across species

Due to species

differences in

PXR ligand binding

site

typical responses to pxr mediated mechanism
Receptor Binding Assays (PXR) – IC50 ~ 5 uM

Transactivation-Reporter Assays (PXR)

Immortalized Cell Lines (Fa2N-4)

Primary Cell Lines (hepatocytes)

Transgenic Animals (hPXR) – 5X increase in mRNA & activity

Clinical Studies (DDI) – 65-98% decreases in AUC

Typical Responses to PXR Mediated Mechanism

Rifampicin

Rifampicin

Untitled

120

100

80

60

Response

40

20

0

-20

-3

-2

-1

0

1

2

Log Concentration(uM)

Fold Increase in CYP3A4 mRNA

slide30

Summary

  • Drug interactions are of great concern to both the pharmaceutical industry and regulatory agencies.
  • Major drug interactions are caused by either inhibition or induction of drug metabolizing enzymes.
  • Models provide numbers that must be placed in context with multiple factors:
    • therapeutic area
    • therapeutic drug concentrations
    • therapeutic index
    • route of administration
    • market competition
    • patient population
slide31

Summary

  • Semi-quantitative predictions of drug interactions
    • many unknown factors
    • human ADME properties in vivo
  • Animal models are not predictive of human interaction potential.
  • Static nature of in vitro systems compared to the dynamic in vivo system
  • Mixtures of interaction mechanisms from the same compound are extremely difficult to predict:
    • reversible + irreversible inhibition
    • inhibition + induction
slide32

Acknowledgments

  • A. David Rodrigues
  • Ken Santone
  • Sean Kim
references
References

Journal Articles

T.D. Bjornsson, et al, The conduct of in vitro and in vivo drug-drug interaction studies: A pharmaceutical research and manufacturers of America perspective, Drug Met. Dispos. 31:815 (2003).

J.H. Lin, Sense and nonsense in the prediction of drug-drug interactions, Curr. Drug Met. 1:305 (2000).

Ito, et al, Prediction of pharmacokinetic alterations caused by drug-drug interactions: Metabolic interaction in the liver, Pharmacol. Rev. 50:387 (1998).

Regulatory Guidance

US FDA CDER, Guidance for industry: Drug metabolism/drug interaction studies in the drug development process: Studies in vitro, www.fda.gov/cder/guidance/clin3.pdf.

European agency for the evaluation of medicinal products, committee for proprietary medicinal products, Note for guidance on the investigation of drug interactions. CPMP/EWP/560/95, www.eudra.org.

Books

Drug Metabolizing Enzymes: Cytochrome P450 and other enzymes in drug discovery and development. Editors J.S. Lee, R. S. Obach, M.B. Fisher, Marcel Dekker, New York (2003).

Drug Drug Interactions, editor A. D. Rodrigues, Marcel Dekker, New York (2002).

Metabolic Drug Interactions, editors R.H. Levy, K.E. Thummel, W.F. Trager, P.D. Hansten, M. Eichelbaum, Lippincot Williams & Wilkines, New York (2000).

Handbook of Drug Metabolism, editor T.F. Woolf, Marcel Dekker, New York (1999).

enzyme kinetics of irreversible inhibition

K3 / k4

= [P]/[EI*]

Partition Ratio =

Enzyme Kinetics of Irreversible Inhibition

K2 * K4

k1

k2

k4

Kinact

=

E + I EI EI’ EI*

K2 + K3 + K4

k-1

k3

E + P

K3 + K4

K-1 + K2

KI

=

*

K2 + K3 + K4

K1

Kinact - the maximal rate of enzyme inactivation

KI - the concentration of inhibitor that gives 50% maximal inhibition

slide36

Assessing Inhibition Potential of

Irreversible Inhibitors

Lambda (l) = [I] * Kinact

[I] + KI

Combining Kinact , KI and Inhibitor Concentration

Lambda is the inactivation rate constant which can be

compared to known irreversible inhibitors with clinically

significant drug interactions.

Mayhew, Hall, Jones (2000) Drug Met. Disp. 28:1031

slide37

Functional Groups For Metabolism-Based P450 Inhibition

Mechanism-based inactivation

Terminal olefins (secobarbital)

Acetylenes (ethinyl estradiol, RU486)

Furans (bergamottins, furafylline)

Thiophene (tienilic acid)

Cyclic amines and N-N functions (phencyclidine)

Quasi-irreversible inhibition

Aryl or alkylmethylenedioxy compounds

Alkyl or aromatic amines (TAO, erythromycin)

1,1-Disubstituted and acyl hydrazines (isoniazid)

slide38

Metabolite - Intermediate (MI) Complex

Quasi-Irreversible Inhibition

Methylene Dioxyphenyl

Derivatives

Characteristic UV max @ 455 nm

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